Catalytic Mechanism and Differential Alarmone Regulation of a Conserved Stringent Nucleosidase

Understanding how bacteria rapidly adapt their metabolism in response to external stimuli is key to addressing the present crisis of antibiotics-resistant infections. In the Gram-negative bacterium Escherichia coli, the universal stringent response is elicited in response to some antibiotics and involves production of the global alarmones, (p)ppGpp, which bind directly to many cellular targets. The nucleosidase PpnN that cleaves nucleotides into 5-phosphate ribose and nucleobase, was shown to be a target of (p)ppGpp and control the delicate balance of bacterial fitness and persistence to fluoroquinolone antibiotics, thus conferring optimal survival strategies to bacteria during antibiotic selective pressure. Although both pppGpp and ppGpp stimulate the enzymatic activity of PpnN, they exert distinct effects on the enzymes cooperativity. The molecular mechanism underlying this subtle difference as well as the precise catalytic mechanism of PpnN, remain obscure. In this study, we provide mechanistic insights into the interaction of PpnN with substrate analogue, reaction products and alarmone molecules, which allows us to understand the catalytic mechanism of this family of nucleosidases and the differential modes of regulation by ppGpp and pppGpp, respectively. Comparison with the homologous LOG proteins involved in cytokinin production in plants reveals an ancient, universal mechanism for cleaving purine monophosphates that bacteria have incorporated regulatory controls through alarmones upon stringent responses. IMPORTANCEThis study explores the distinct manners that the stringent alarmones pppGpp and ppGpp interact with the nucleosidase PpnN, which diverge from their typical, consistent effects on other target proteins. Furthermore, PpnN plays a key role in balancing bacterial fitness and tolerance to antibiotics, yet its catalytic mechanism has remained unclear. Through a combination of structural biology, molecular simulations, biochemistry, mutagenesis, and physiological analyses, this research uncovers the mechanistic differences in how both alarmones differentially impact enzyme cooperativity of PpnN and activation under stress conditions. Additionally, by analysing structures of PpnN complexed with substrate analogues, reaction products, and alarmones, as well as conducting bioinformatic comparisons, we propose a conserved catalytic mechanism shared with its homologue, LOG, a protein involved in cytokinin signalling - a critical growth hormone in plants. These insights underscore the nuanced regulatory roles of alarmones and PpnN/LOG group of nucleosidases, highlighting their evolutionary diversification to meet the varied environmental survival needs across organisms.